体外筛选炭疽杆菌芽孢适配子的研究与初步应用
摘要
SELEX技术(Systematic evolution of ligands by exponentialenrichment)即指数扩增配体的系统进化技术，是90年代初建立的一种体外文库筛选方法。SELEX技术的基本思想是合成一个两端为固定序列，中间为随机序列的单链寡核苷酸库(RNA或DNA)，库中序列的多样性可反映为二级结构的多样性；将它与靶物质混合，形成靶物质—寡核苷酸的复合物，去除未与靶物质结合的核酸，分离与靶物质结合的核酸分子，并以此类核酸分子为模板由固定序列为引物进行PCR扩增，扩增产物再进行下一轮的筛选过程；通过多轮的筛选与扩增，一些与靶物质不结合或与靶物质只有低亲和力的寡核苷酸被去除，而与靶物质有较高亲和力的“适配子”(Aptamer)将逐渐得到富集。这一方法的原理简单，操作简便易行，一经问世，便得到了广泛的应用。适配子具有亲和力高且特异性好等特点，而且易于标准化，目前正在成为一种新的诊断试剂。目前，已有研究者将凝血酶的适配子成功地固化在光纤传感器上，用以检测凝血酶，这一工作为生物战剂传感器的研制提示了新的方向。国外有利用SELEX技术筛选炭疽杆菌芽孢适配子的报道，但其使用的是高压灭菌处理后的芽孢，而且没有完成适配子特异性、亲和力以及结构与功能的考察，在这一方面还有许多工作急待进行。国内有关SELEX技术的研究起步较晚，文献报道较少。
本研究工作将系统地进行炭疽杆菌芽孢适配子的研究，并基于适配子的特性，建立一种以尼龙膜为载体，炭疽芽孢适配子为检测分子来检测炭疽芽孢的方法；此外，由于许多生物战剂的单克隆抗体研制存在困难，本研究可为解决这一难题提供新的思路。我们一直期望能将SELEX这一新技术应用到微生物检测的研究工作中。适配子是一段寡核苷酸，其稳定性优于单克隆抗体，已有实验证明SELEX技术可以筛选到特异性和亲和力都强于单克隆抗体的适配子；而适配子与靶物质的结合是结构依赖性，而不是序列依赖性的，因此不需杂交等步骤；此外适
配于的筛选是一种体外筛选，不涉及免疫过程，适用范困广。
基于上述考虑，结合国外的技术进展和自身工作的实际需要，我们
拟定了这一课题，目的不仅在于以炭疽杆菌的芽抱作为筛选模式，筛选
到炭疽芽抱特异的适配子，将筛选到的适配子作结构与功能研究，并将
亲和性较好的适配子作为检测炭疽芽抱的检测分子；还期望在我们实验
室建立起行之有效的SELEX技术，用于单克隆抗体制备存在困难的其它
生物战剂，为生物战剂的快速侦检开辟新的思路。
研究结果显示：在体外构建的两端为固定序列，中I’riJ随机区域为
35个核昔酸序列，其全长为78个核昔酸的随机寡核昔酸文库的基础卜，
用炭疽秆菌疫茵株 A．161｛的芽抱为靶物质，在含有 2价 Mg‘”存在的情况
下，按SSDNA：炭疽芽抱＝l…g：1．5X108混合，经过了十／轮“吸附－
漂洗－洗脱－扩增”的SELEX过程，同时，每三轮的筛选产物用蜡样芽
抱杆菌芽抱进行反筛，以提高筛选的特异性。筛选产物经扩增后在 15％
的聚丙烯酚胺凝胶电泳图中显示，随着筛选轮数的增加，PCR扩增电
泳条带逐渐单纯。为了提高产物的忠实性，PCR扩增的优化条件为：
采用热启动法扩增可以稳定地获得较好的扩增结果；退火温度在58OC
～68C、循环数为20～22时，有预期的扩增带。将每轮的筛选产物用标
有生物素的引物PCR扩增后，与炭疽芽抱进行结合实验，TMB显色系
统提示，第十八轮筛选的炭疽芽抱适配子库与芽抱结合后显色O＊值
比第一轮的O＊值提高了37 fg以上，在第六轮筛选时，出现了O＊值
降低，这种现象的产生是山于筛选压”（选择／进化压）的影响，使得筛
选库中所期望得到的寡核昔酸片段的特异性增强。在考察筛选库的特异
性时，将高压灭菌后的炭疽芽抱和筛选用炭疽芽泡约 IXIO’分别与第十
八轮的筛选库巾g作结合实验，检测的0．D值分别是0．193和 1．1H，
说明了筛选产物对靶物质的特异性较强。考虑到芽抱是一个多表位的靶
物质，是个混合靶物质的筛选过程，筛选的适配子是多样的，我们将筛
选库进行了单链和双链（加热与不加热）DNA与炭疽芽抱的结合实验。
结果显示，适配子与芽抱结合以单链为主，双链也有结合。
在经过了成功的十八轮SELEX筛选后，将筛选产物－寡核昔酸片段
进行了克隆，在500个克隆子中随机选取79个克隆子进行了测序，将
测序结果按碱基多少分为A、BVH群，AJty的54条与预期片段大小一致，
一10 一
少数片段在3’端固定区有个别碱基突变山群为25条大小不等的片段。
可分为两个亚群，BI群为12条随机序列区域短于所期望长度的片段，
并在随机区域或 3’端引物区域有碱基突变；BZ群为＊ 条长于预期长
度的片段，主要表现在3’端引物序列的重复出现。造成短于彬］望片段
长短的原因可能是山于跳跃PCR的影响；长于期望片段长度的原因PJ‘能
是低温下，引物与随机区域退火延伸所致。将测序序列（适配－f）计算
机分析软件分析测序序列的保守
SELEX (System Evolution of Ligands by Exponential enrichment), which is a universally-applied method for spanning of aptamer from oligonucleotide pool as potential pharmaceutical and diagnostic agents, has been under development for more than a decade. SELEX technology provides an excellent opportunity to search for oligonucleotides binding to target and has an advantage over other known methods for ligands discovery, such as the small molecule combinatorial chemistry and phage-displayed antibody technique. SELEX can be essentially applied to any target and generate a large number of lead compounds very quickly, reducing much work and saving much time before animal testing. SELEX begins, with a large population of single-stranded nucleic acid molecules that is challenged with a specific task. Usually, the task is to bind to a purified protein, yet many other tasks have been successfully selected, including cell binding, peptide binding, small molecule binding, nucleic acid binding and catalysis of a variety
of chemical reactions. A successful SELEX experiment requires the determination of a method for partitioning those nucleic acid molecules that have performed the task from those that have not. A pool of randomized RNA or single stranded DNA sequences was selected against
interested targets. Several rounds of spanning lead to winning sequences with specific structure called 'aptamers', which are single-stranded nucleic acids that perform a specific function. Structures of aptamer complexes reveal the key molecular interactions conferring specificity to the aptamer-ligand association, including the precise stacking of flat moieties, specific hydrogen binding, and molecular shape complementarities. The oligonucleotide aptamers selected by SELEX process are superior in terms of higher affinity, specificity and stability. This technology has found a wide range of applications, yet little work has been reported on development of SELEX aptamers to interact infectious disease agents or their epitopes for either diagnosis or potential therapy with the exceptions of SELEX aptamers specific for HIV components and a selection model for autoclaved anthrax spores. And anti-Bacillus anthracis spores aptamers' research related with structures and functions is remaining urgently.
In the present report, our aim is not only to establish a mold taking selection as anthrax spores for a target, but also to study the relationship between structures and affinities of SELEXed DNA aptamers to Bacillus anthracis spores. Meanwhile choosing the higher affinity aptamer as the detection molecules for Bacillus anthracis spores, we open a new throughway for a rapid detection of the pathogen microbiology used in the war.
The research results showed that a custom synthesized 78mer random oligonucleotides DNA library (ca. 10l4~l5types of different DNA), containing 35 random nucleotides flanked by primer annealing sites, was chemosynthesized, and was subjected to 18 rounds of selection of 'binding-washing-separation-PCR amplifying' by using SELEX method against spores of Bacillus anthracis vaccine strain A.16R. There were totally 18 rounds of selection and after each 3 rounds a counter-selection was performed. Counter-selection was binding reaction between the PCR product from each three round and the microcentrifuge tube used in the selection process and Bacillus cereus spores to getting rid of the
components bound to the tube and those unspecific to anthrax spores. 15%PAGE electrophoresis analysis of PCR products from different selection rounds were showed that electrophoresis bands of the expected size got compact with the progress of SELEX rounds. An optimization strategy was carried out with annealing at 58-68癈 and 20-22 cycles to ensure complete elongation of the products. 'Hot star1 strategy, with adding Tap DNA polymerase at penetration temperature, was employed in the experiment. Selected products in each round were amplified through PCR with biotin-modified primers. The amplified product in each round to the spores was visu
本研究工作将系统地进行炭疽杆菌芽孢适配子的研究，并基于适配子的特性，建立一种以尼龙膜为载体，炭疽芽孢适配子为检测分子来检测炭疽芽孢的方法；此外，由于许多生物战剂的单克隆抗体研制存在困难，本研究可为解决这一难题提供新的思路。我们一直期望能将SELEX这一新技术应用到微生物检测的研究工作中。适配子是一段寡核苷酸，其稳定性优于单克隆抗体，已有实验证明SELEX技术可以筛选到特异性和亲和力都强于单克隆抗体的适配子；而适配子与靶物质的结合是结构依赖性，而不是序列依赖性的，因此不需杂交等步骤；此外适
配于的筛选是一种体外筛选，不涉及免疫过程，适用范困广。
基于上述考虑，结合国外的技术进展和自身工作的实际需要，我们
拟定了这一课题，目的不仅在于以炭疽杆菌的芽抱作为筛选模式，筛选
到炭疽芽抱特异的适配子，将筛选到的适配子作结构与功能研究，并将
亲和性较好的适配子作为检测炭疽芽抱的检测分子；还期望在我们实验
室建立起行之有效的SELEX技术，用于单克隆抗体制备存在困难的其它
生物战剂，为生物战剂的快速侦检开辟新的思路。
研究结果显示：在体外构建的两端为固定序列，中I’riJ随机区域为
35个核昔酸序列，其全长为78个核昔酸的随机寡核昔酸文库的基础卜，
用炭疽秆菌疫茵株 A．161｛的芽抱为靶物质，在含有 2价 Mg‘”存在的情况
下，按SSDNA：炭疽芽抱＝l…g：1．5X108混合，经过了十／轮“吸附－
漂洗－洗脱－扩增”的SELEX过程，同时，每三轮的筛选产物用蜡样芽
抱杆菌芽抱进行反筛，以提高筛选的特异性。筛选产物经扩增后在 15％
的聚丙烯酚胺凝胶电泳图中显示，随着筛选轮数的增加，PCR扩增电
泳条带逐渐单纯。为了提高产物的忠实性，PCR扩增的优化条件为：
采用热启动法扩增可以稳定地获得较好的扩增结果；退火温度在58OC
～68C、循环数为20～22时，有预期的扩增带。将每轮的筛选产物用标
有生物素的引物PCR扩增后，与炭疽芽抱进行结合实验，TMB显色系
统提示，第十八轮筛选的炭疽芽抱适配子库与芽抱结合后显色O＊值
比第一轮的O＊值提高了37 fg以上，在第六轮筛选时，出现了O＊值
降低，这种现象的产生是山于筛选压”（选择／进化压）的影响，使得筛
选库中所期望得到的寡核昔酸片段的特异性增强。在考察筛选库的特异
性时，将高压灭菌后的炭疽芽抱和筛选用炭疽芽泡约 IXIO’分别与第十
八轮的筛选库巾g作结合实验，检测的0．D值分别是0．193和 1．1H，
说明了筛选产物对靶物质的特异性较强。考虑到芽抱是一个多表位的靶
物质，是个混合靶物质的筛选过程，筛选的适配子是多样的，我们将筛
选库进行了单链和双链（加热与不加热）DNA与炭疽芽抱的结合实验。
结果显示，适配子与芽抱结合以单链为主，双链也有结合。
在经过了成功的十八轮SELEX筛选后，将筛选产物－寡核昔酸片段
进行了克隆，在500个克隆子中随机选取79个克隆子进行了测序，将
测序结果按碱基多少分为A、BVH群，AJty的54条与预期片段大小一致，
一10 一
少数片段在3’端固定区有个别碱基突变山群为25条大小不等的片段。
可分为两个亚群，BI群为12条随机序列区域短于所期望长度的片段，
并在随机区域或 3’端引物区域有碱基突变；BZ群为＊ 条长于预期长
度的片段，主要表现在3’端引物序列的重复出现。造成短于彬］望片段
长短的原因可能是山于跳跃PCR的影响；长于期望片段长度的原因PJ‘能
是低温下，引物与随机区域退火延伸所致。将测序序列（适配－f）计算
机分析软件分析测序序列的保守
SELEX (System Evolution of Ligands by Exponential enrichment), which is a universally-applied method for spanning of aptamer from oligonucleotide pool as potential pharmaceutical and diagnostic agents, has been under development for more than a decade. SELEX technology provides an excellent opportunity to search for oligonucleotides binding to target and has an advantage over other known methods for ligands discovery, such as the small molecule combinatorial chemistry and phage-displayed antibody technique. SELEX can be essentially applied to any target and generate a large number of lead compounds very quickly, reducing much work and saving much time before animal testing. SELEX begins, with a large population of single-stranded nucleic acid molecules that is challenged with a specific task. Usually, the task is to bind to a purified protein, yet many other tasks have been successfully selected, including cell binding, peptide binding, small molecule binding, nucleic acid binding and catalysis of a variety
of chemical reactions. A successful SELEX experiment requires the determination of a method for partitioning those nucleic acid molecules that have performed the task from those that have not. A pool of randomized RNA or single stranded DNA sequences was selected against
interested targets. Several rounds of spanning lead to winning sequences with specific structure called 'aptamers', which are single-stranded nucleic acids that perform a specific function. Structures of aptamer complexes reveal the key molecular interactions conferring specificity to the aptamer-ligand association, including the precise stacking of flat moieties, specific hydrogen binding, and molecular shape complementarities. The oligonucleotide aptamers selected by SELEX process are superior in terms of higher affinity, specificity and stability. This technology has found a wide range of applications, yet little work has been reported on development of SELEX aptamers to interact infectious disease agents or their epitopes for either diagnosis or potential therapy with the exceptions of SELEX aptamers specific for HIV components and a selection model for autoclaved anthrax spores. And anti-Bacillus anthracis spores aptamers' research related with structures and functions is remaining urgently.
In the present report, our aim is not only to establish a mold taking selection as anthrax spores for a target, but also to study the relationship between structures and affinities of SELEXed DNA aptamers to Bacillus anthracis spores. Meanwhile choosing the higher affinity aptamer as the detection molecules for Bacillus anthracis spores, we open a new throughway for a rapid detection of the pathogen microbiology used in the war.
The research results showed that a custom synthesized 78mer random oligonucleotides DNA library (ca. 10l4~l5types of different DNA), containing 35 random nucleotides flanked by primer annealing sites, was chemosynthesized, and was subjected to 18 rounds of selection of 'binding-washing-separation-PCR amplifying' by using SELEX method against spores of Bacillus anthracis vaccine strain A.16R. There were totally 18 rounds of selection and after each 3 rounds a counter-selection was performed. Counter-selection was binding reaction between the PCR product from each three round and the microcentrifuge tube used in the selection process and Bacillus cereus spores to getting rid of the
components bound to the tube and those unspecific to anthrax spores. 15%PAGE electrophoresis analysis of PCR products from different selection rounds were showed that electrophoresis bands of the expected size got compact with the progress of SELEX rounds. An optimization strategy was carried out with annealing at 58-68癈 and 20-22 cycles to ensure complete elongation of the products. 'Hot star1 strategy, with adding Tap DNA polymerase at penetration temperature, was employed in the experiment. Selected products in each round were amplified through PCR with biotin-modified primers. The amplified product in each round to the spores was visu
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